Methods for processing microscopic samples or objects by laser microdissection have been in existence since the mid-1970s and have continuously been developed further since then.
In laser microdissection, cells, tissue regions, etc. can be isolated from a tissue complex and obtained as dissectates. A particular advantage of laser microdissection is that the tissue is in contact for a short time with the laser beam, by which the tissue next to the laser beam is scarcely altered. The dissectates can specifically be obtained in different ways.
For example, a dissectate can be isolated from a sample by means of an infrared or ultraviolet laser beam and falls into a suitable dissectate collector under the influence of gravity. The dissectate can in this case also be cut out of the sample together with an adhering membrane.
Another method is laser capture microdissection. In that method, a thermoplastic membrane, which may also be connected to a reaction vessel, is heated by means of a corresponding laser beam. The membrane melts with the desired region of the object and can be removed in a subsequent step. A further alternative consists in sticking the dissectate to a cap of a dissectate collector by means of the laser beam. Inverted microscope systems for laser microdissection are also known.
Known microscope systems for laser microdissection have a reflected light device, into the beam path of which a laser beam is coupled. The laser beam is focused by the particular microscope objective used onto the sample, which is lying on a microscope stage which can be displaced automatically by means of a motor. A cut line is produced, for example, by displacing the microscope stage during cutting in order to move the sample relative to the fixed laser beam. However, this has the disadvantage, inter alia, that the sample cannot be observed properly during production of the cut line because it moves in the field of view.
More advantageous laser microdissection systems therefore have laser scanning devices which are configured correspondingly to displace the laser beam or the point of impact thereof on the sample, which is then fixed. Such laser microdissection systems also have particular advantages in the context of the present invention. A particularly advantageous laser microdissection system of the type mentioned, which has a laser scanning device with wedge prisms, is described in EP 1 276 586 B1.
In both cases, that is to say both in laser microdissection systems in which the microscope stage is displaced and in laser microdissection systems which have a laser scanning device, the operation is generally carried out using pulsed lasers, a hole being produced in the sample by each laser pulse. A cut line forms by stringing such holes together, optionally with overlapping.
In microscopic examination methods, for example in medical diagnostics, magnifying digital optical imaging devices, in particular slide scanners, are frequently used in addition to microscopes in the narrower sense. Slide scanners are used to produce partial or complete images of slides with microscopic samples applied thereto, which images can then be evaluated on a screen and/or stored. The evaluation can also be made partially or fully automatically, for example using pattern recognition methods by means of which, for example, pathologically altered cell or tissue types can be identified. A slide scanner thus permits examination or diagnosis on the basis of digital images of samples without the direct use of a microscope. Slide scanners have the advantage of a high throughput and allow a large number of samples to be processed largely automatically.
If cell or tissue types requiring additional molecular-biological and/or biochemical investigation are detected in digital images of a corresponding digital optical imaging device, for example a slide scanner, corresponding regions of a microscopic sample can be processed in a laser microdissection system, that is to say can be cut out of the sample in such a system.
However, the subsequent processing by laser microdissection of samples which have previously been examined by means of magnifying digital optical imaging devices, for example the mentioned slide scanners, is conventionally found to be highly complex. In particular, it is conventionally not possible to define specific regions on the basis of a digital image of a microscopic sample and process exactly the same regions of the same object or slide in a laser microdissection system.
Instead, the prior art in this context is the production of serial sections, as disclosed, for example, in US 2012/0045790 A1. In this case, two adjacent thin tissue sections are prepared from a tissue block, for example by means of a microtome, and treated differently. The first section is subjected to a standard treatment and subsequent production of a corresponding image. Staining of the sample and digitization in a slide scanner are carried out, for example. On the basis of this section, a pathologist selects sample regions for examination in a laser microdissection system. Corresponding region information is stored in a laboratory information system. The second section is supplied in parallel to the laser microdissection system. In the laser microdissection system, this second section is then processed on the basis of the region selection made in respect of the first section. Digital image overlay programs, which are comparatively slow and are not sufficiently accurate for reliable results, may also be used in this connection.